1. Field of the Invention
The present invention is directed to a process for selectively extracting cocoa procyanidins according to their degree of polymerization, using particular sequences of solvents. In particular, the present invention is directed to a process for selectively extracting individual oligomers or small groups of oligomers from a mixture of cocoa procyanidins, using n-butyl acetate as the first extraction solvent.
It is known that individual flavan-3-ols, such as catechin, epicatechin and cocoa procyanidin oligomers, exhibit distinct properties and have distinct applications for human and animal use. Selectively extracting specific monomers or oligomers on the basis of degree of polymerization will allow for more targeted and efficacious use thereof, in pharmacological applications, as well as in food-grade products.
2. Discussion of the Related Art
Proanthocyanidins, the oligomers and polymers of flavan-3-ols, are the second most abundant natural plant phenol after lignin. The flavan-3-ol subunits are linked primarily through a carbon-carbon bond from the 4 position of one subunit to the 8 position of another subunit (C4→C8), and to a lesser extent through C4→C6 linkage.
Procyanidins represent the largest class of proanthocyanidins. Gu et al. showed that out of 41 foods found to contain proanthocyanidins, 27 contained procyanidins. (J. Agric. and Food Chem. 51 (2003) 7513). Procyanidins typically consist of (−)-epicatechin, (+)-epicatechin, (−)-catechin and/or (+)-catechin subunits.
Procyanidins include B-type and A-type proanthocyanidins. In B-type proanthocyanidins, the monomeric subunits (catechin, epicatechin) are connected via interflavan linkages of C4→C6 and/or C4→C8. Oligomers with exclusively C4→C8 linkages are linear, while the presence of at least one C4→C6 bond results in a branched oligomer. By contrast, A-type proanthocyanidins are doubly-linked oligomers, containing linkages C2-O-C7 and C4→C6 or C4→C8.
The molecular weight of proanthocyanidins typically is expressed as degree of polymerization (DP), and individual oligomers are commonly referred to as dimers, trimers, etc.
It is known that individual procyanidin oligomers present specific characteristics and potential benefits for use in humans and animals. For example, Tempesta discloses that procyanidin oligomers having a degree of polymerization (DP) of 2-11 possess significant antiviral activity, and are useful in treating warm-blooded animals, including humans, infected with paramyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such as Herpes Simplex virus. (U.S. Pat. No. 5,211,944). Romanczyk Jr., et al. disclose antineoplastic compositions comprising procyanidin oligomers having a DP of 3-11 together with a suitable carrier. (U.S. Pat. No. 5,554,645). Romanezyk, Jr. et al. also disclose that procyanidin oligomers having a DP of 5-12 are useful as antioxidants. (U.S. Pat. No. 5,891,905). Schmitz et al. disclose the use of cocoa procyanidin oligomers (DP of 2-18) together with acetylsalicylic acid as anti-platelet therapy. (U.S. Pat. No. 6,524,630).
Significant improvements in the separation and resolution of procyanidin oligomers have been achieved. (See, Rigaud et al., Chromatogr. 654 (1993) 179; Cheynier et al., Methods in Enzymology 299 (1999); Natsume et al., Biosci. Biotechnol. Biochem. 64 (2000) 2581). Resolution of procyanidin oligomers up to the pentamer (DP=5) has been obtained. Hammerstone et al. disclosed modifications of this method, leading to improvements in resolution of monomers through the nonamers in the analysis of unfermented cacao seeds. (J. Agric. and Food Chem. 47 (1999) 490). Gu et al. disclosed still further improvements, leading to the elution of a polymer peak (DP>10), as well as enhancement in overall peak shape and resolution. (J. Agric. and Food Chem. 50 (2002) 4852).
It would be desirable to be able to maximize the loading capacity of cocoa procyanidins onto a preparative scale HPLC system, so that greater quantities of desired specific monomers and/or oligomers could be eluted for further study or use. Theoretically, the loading capacity of a preparative scale HPLC column (300×55 mm, 100 μm) is approximately 4 g of material. However, cocoa procyanidin extracts present solubility problems that limit the maximum loading capacity to approximately 400 mg, of which only approximately 41% are flavonoids.
It is expected that a significant increase in sample loading (flavanol) content can be achieved via selective extraction of individual or small groups of oligomers from a polyphenol mixture, prior to insertion onto an HPLC column. Accordingly, what is needed is a method for selectively extracting other desired cocoa procyanidin oligomers from an aqueous extract of cocoa polyphenols on the basis of degree of polymerization.